SCIENTIFIC ABSTRACT Accumulating evidence suggests that disruption of circadian rhythm by shift-work increases the risk of breast, prostate, and colon cancers, prompting the International Agency for Research on Cancer to classify shift-work as a probable human carcinogen. Identification and validation of mechanistically-based biomarkers is essential to the development of preventive strategies for individuals who may be at elevated risk of breast cancer due to shift-work. We've demonstrated that carcinogen N-Nitroso-N-methylurea (NMU) reduced intracellular circadian cycling and levels of NAD+/NADH and decreased SIRT1 activity, disrupting circadian expression of core circadian genes (CGs) (e.g., Per2) and circadian controlled genes (CCGs), including DNA damage response and repair (DDRR) genes. In contrast, chemopreventive methylselenocysteine (MSC) counteracted the inhibitory effects of NMU on NAD+/NADH and SIRT1 activity and restored the circadian expression of these genes during early stages of carcinogenesis, leading to 63% reduction of mammary tumor incidence in rats. Consistently, accumulating evidence indicates that modulation of NAD+/NADH levels by pharmacological inhibitors or by NAD+ precursors alter mammary tumor growth and metastasis in animals. We further revealed that jet-lag also disrupted the rhythmic expression of Per2 and DDRR, and accelerated mammary tumorigenesis and metastasis in rodents. Therefore, we posit that NAD+-dependent SIRT1 activity functions as a central integrator of cellular circadian responses to environmental stressors, and hence could serve as novel mechanistically-linked biomarkers of increased risk of breast cancer due to circadian disruption in shift workers. We also demonstrated that working night- vs day-shift significantly changed Per2 gene expression pattern in peripheral blood samples, as we observed in mammary glands of rodents. Therefore, we hypothesize that shift-work associated disruption of circadian rhythm reduces intracellular rhythms and levels of NAD+/NADH and SIRT1 activity, which may in turn increase the risk of breast cancer by inducing abnormal expression of CCGs, including most DDRR. To test our hypothesis, and to identify and validate novel mechanistic biomarkers of circadian disruption for use in future intervention trials in shift workers, we propose to determine intracellular rhythms and levels of the NAD+/NADH and SIRT1 activity in peripheral blood mononuclear cells (PBMCs) of both day-shift and night-shift workers over 24 hr. We also propose to validate NAD+/NADH and SIRT1 activity in PBMCs as biomarkers of disrupted circadian rhythm in night-shift workers under real-working conditions. These studies will provide baseline and circadian data on NAD+/NADH and SIRT1 activity in shift workers, and we will evaluate them as biomarkers of disrupted circadian rhythm that may increase the risk of breast cancer. These biomarkers can then be used in future studies to explore preventive strategies with dietary agents in women at elevated risk of breast cancer due to shift-work.